PROJECT SUMMARY/ABSTRACT 4D fluorescence microscopy is considered the workhorse for biomedical research. It provides a window into the spatially complex, temporally evolving physiology of living specimens with high specificity. The knowledge gained from the 4D space-time data has also facilitated the discovery of informative biomarkers that can be used for the early detection of diseases and for the development of drugs directed at new therapeutic targets. However, many cellular structures and events are below the spatial resolution limit of traditional fluorescence microscope and happen on sub-second time scale, putting them beyond our ability to investigate in greater detail. In addition, observing the fast evolving dynamic process over the entire 3D volume involves inevitable tradeoffs on spatial resolution, temporal resolution, sample-induced aberrations, and phototoxicity. This last point is especially important for 4D live-cell imaging, where phototoxicity caused by the high level of free radicals would alter the physiological state of living specimens. In this project, we propose to develop a super-resolution imaging method, termed fluorescence ptychographic microscopy, for 4D live cell imaging with high spatiotemporal resolution and reduced phototoxicity. The proposed approach will be built upon the structured illumination microscopy (SIM) technique that combines multiple acquisitions under sinusoidal illumination patterns for super-resolution imaging. We aim to significantly shorten the acquisition time of the SIM approach by using a modified ptychographic recovery procedure developed in the PI?s lab. More importantly, we will also develop a ptychographic procedure that is able to correct for the unknown distortions of the sinusoidal patterns and compensate for the sample-induce wavefront aberrations. If successful, the proposed imaging procedure would provide a turnkey solution for 4D fluorescence microscopy with sub-diffraction spatial resolution and sub-second temporal resolution, with minimum invasiveness for the living specimen, and is able to compensate for sample-induced aberrations. Our long term goal is to translate advanced imaging technologies for the broad biomedical and clinical communities.